Production of recombinant bmp-2

ABSTRACT

In order to recombinantly prepare a biologically active protein of the TGF-β superfamily, a protein, whose amino terminus consists of the pro sequence of a protein of the TGF-β superfamily, or parts thereof, to which the mature domain of this protein or of another protein of TGF-β superfamily which exhibits at least 35% homology with mature BMP-2 is attached, is expressed in prokaryotes under conditions in which at least a part of the protein is obtained in the form of inclusion bodies, the inclusion bodies are isolated and solubilized under denaturing conditions, the denatured, monomeric and biologically inactive protein which has been solubilized from the inclusion bodies is renatured, with folding and dimerization to give the soluble, biologically active conformation and, where appropriate, after the renaturation, the mature protein is released proteolytically from its pro form.

DESCRIPTION

[0001] The present invention relates to a process for obtainingbiologically active drBMP-2 (dr: digit removed), and homologousproteins, by renaturing denatured, biologically inactive proBMP-2 and,where appropriate, subsequently eliminating the propeptide, with therenatured pro form of the protein already being biologically active, toproBMP-2 as a novel product and to its use for producing a compositionfor promoting bone growth.

[0002] BMP-2 belongs to the family of bone morphogenetic proteins(BMPs). These proteins are bone growth factors whose cloning andcharacterization were achieved for the first time in 1988. Thecharacteristic property of these proteins is the ability to induceendochondral bone neoformation [Wozney et al., Science 242 (1988),1528-1534].

[0003] Structurally, BMP-2 belongs, together with more than 30 otherproteins, to the TGF-β superfamily, the members of which exhibit a veryhigh degree of structural similarity to each other although theirsequence identity is in some cases less than 30% [Griffith et al., Proc.Natl. Acad. Sci. USA 93 (1996), 878-883]. The essential structuralfeature of these proteins is what is termed the “cystine knot”. This isformed by four antiparallel β-pleated sheet strands, two of which are ineach case linked to each other by way of disulfide bonds. In connectionwith this, two cystines form, together with the peptide backbone, a ringthrough which a third disulfide bridge extends.

[0004] β-Pleated sheets are the dominant secondary structural element inthese proteins. However, in these proteins, they are longer and moretightly twisted than in other β-pleated sheet-rich proteins. Because ofthe structural relatedness of the proteins of the TGF-β family to eachother, we have postulated that these proteins also assume their nativeconformation, that is fold, in a similar manner.

[0005] BMPs and related proteins, such as DPP (decapentaplegic,Drosophila), play an important role in the embryonic development ofhigher organisms. Defects in BMP genes or BMP receptor genes, or theirregulation, lead to what are in some cases severe developmentaldisturbances, such as fibrodysplasia ossificans progressiva [Kaplan etal., Calcif. Tissue Int. 47 (1990), 117-125; Rao et al., Hum. Genet. 90(1992), 299-302; Shafritz et al., N. Engl. J. Med. 335 (1996), 555-561]and dentinogenesis imperfecta [Tabas et al., Clin. Orthop. 293 (1993),310-316]. Mouse BMP deletion mutants die as early as in the embryonicstage or shortly after birth and exhibit malformations of the skeletonand a very wide variety of organs, such as the heart, kidneys and eyes[Dudley et al., Genes Dev. 9 (1995), 2795-2807; Zhang and Bradley,Development 122 (1996), 2977-2986].

[0006] BMPs and similar growth factors are only suitable for treatingsystemic diseases, such as osteoporosis, under certain circumstancessince they can elicit non-tolerable side effects, such as tumorpromotion. Rather, BMPs, in particular BMP-2, are particularly suitablefor locally restricted applications. These include, inter alia, spinalfusions in connection with degenerative diseases of the vertebralcolumn, the neoformation of cranial bones, maxillofacial surgicalinterventions and the treatment of complicated fractures of the bones ofthe extremities. In the case of these applications, the positive effectof rhBMP-2 (rh—recombinant human) on bone neoformation, and consequentlyon the healing process, has already been impressively demonstrated inseveral animal models [Lane et al., Clin. Orthop, 361 (1999), 216-227;Marden et al., J. Biomed. Mat. Res. 28 (1994), 1127-1138; Mayer et al.,Plast. Recontr. Surg. 98 (1996), 247-259; Sandhu and Boden, Orthop.Clin. North Am. 29 (1998), 621-631; Toriumi et al., Arch. Otolyryngol.Head Neck Surg. 117 (1991), 1101-1112; Yasko et al., J. Bone Joint Surg.(Am.) 74 (1992), 659-670]. In the case of spinal fusion operations, thefirst clinical studies are even available [Boyne et al., Int. J.Periodontics Restorative Dent. 17 (1997), 11-25; Howell et al., Int. J.Periodontics Restorative Dent 17 (1997), 124-139]. It is likewiseappropriate to use rhBMP-2 for fixing artificial joints or screwfittings, as are used, for example, for stabilizing fractures, in thesurrounding bone tissue.

[0007] Apart from the studies which are still lacking, and also thesestudies themselves, it is, in particular, the high costs of obtainingthe proteins which stand in the way of using BMPs routinely in clinicalpractice. The process which is described in the present inventionenables biologically active BMP-2, as well as related BMPs, to beprepared recombinantly, economically and at high purity, with littletechnical input and yields which have previously not been thoughtpossible.

[0008] Human BMP-2 is synthesized in the body in the form of apreproprotein of 396 amino acids in length. The signal peptide (presequence) serves in vivo for transporting the nascent polypeptide chaininto the endoplasmatic reticulum (ER). After it has been imported intothe ER, the protein folds into its native conformation, the disulfidebridges (Cys²⁹⁶-Cys³⁶¹, Cys³²⁵-Cys³⁹³, Cys³²⁹-Cys³⁹⁵, intermolecular:Cys³⁶⁰-Cys³⁶⁰), are formed and posttranslational modifications takeplace. The latter include the glycosylation of asparagine residues andthe, partially incomplete, elimination of the propeptide [Israel et al.,Growth Factors 7 (1992), 139-150]. The 114 C-terminal amino acids(Gln²⁸³-Arg³⁹⁶) of the precursor protein form the mature BMP-2, whosebiologically active form is a disulfide-bridged (Cys³⁶⁰-Cys³⁶⁰)homodimer.

[0009] Isolating BMP-2 from bone tissue requires substantial input andonly results in low yields (40 μg of BMP mixture from 40 kg of bovinebone powder) [Wozney et al., Science 242 (1988), 1528-1534].Furthermore, isolation from human bone material is ethically dubious andinvolves the danger of contamination with pathogens (prions, HCV, etc.).The use of homologous proteins from pigs or cattle is likewise riskyfrom the immunological point of view.

[0010] Preference is therefore given to preparing BMP-2 recombinantly.There are two primary possibilities for doing this. In the first place,the protein can be obtained in eukaryotic expression systems [Wang etal., Proc. Natl. Acad. Sci. USA 87 (1990), 2220-2224]. Disadvantages ofthis approach are the relatively low quantities of recombinant protein(25 μg of partly purified BMP-2 per 1 l of medium [Wozney et al.,Science 242 (1988), 1528-1534]) and the relatively high degree oftechnical input and the costs resulting from this.

[0011] The second route involves using prokaryotic expression systems.Their advantages lie in simple technical manipulation, low costs and theisolation of very high quantities of protein. However, in contrast toeukaryotes, bacteria are unable to process the precursor proteincorrectly. For this reason, the method which has thus far been used isrestricted to expressing the mature domain of BMP-2 (Gln²⁸³-Arg³⁹⁶). Inbacteria, this domain accrues in an insoluble, biologically inactiveform since, as a result of the reducing conditions in the cytosol, interalia, it is not possible for the disulfide bridges of the protein to beformed. These insoluble protein aggregates are termed inclusion bodies(IBs), whose isolation and purification has been described, for example,by Marston [Biochem. J. 240 (1986), 1-12].

[0012] The method of Ruppert et al., [Eur. J. Biochem. 237 (1996),295-302] for renaturing mature BMP-2 is based on U.S. Pat. No. 5,650,494[Cerletti et al., 1997], which relates to TGF-β-like proteins, whichalso include BMP-2. The method involves renaturing the monomeric,denatured mature form of TGF-β-like proteins in the presence of a milddetergent, to give the biologically active conformation. In the case ofBMP-2, this only resulted in yields of 0.2 mg of active protein per 1 gof cells being obtained [Ruppert et al., Eur. J. Biochem. 237 (1996),295-302].

[0013] However, using the present invention, it is possible to obtain ayield of active protein which is more than 25 times as high, based onthe induced cell mass. Indeed, the yield is about three orders of sizehigher than that achieved when expressing in eukaryotic cells.

[0014] Because of the complicated disulfide pattern (cystine knot), theyields are very low when renaturing mature BMPs which are expressedrecombinantly in E. coli. The present invention is based on thehypothesis that the BMP-2 propeptide, like the propeptides of homologousproteins as well, is partly responsible for the correct folding of themature domain.

[0015] The process according to the invention for recombinantlypreparing a biologically active protein of the TGF-β superfamily ischaracterized in that (a) a protein, whose amino-terminus consists ofthe pro sequence of a protein of the TGF-β superfamily, or partsthereof, to which the mature domain of this protein, or of anotherprotein of the TGF-β superfamily which exhibits at least 35% homologywith mature BMP-2, is attached, is expressed in prokaryotes underconditions in which at least a part of the protein is obtained in theform of inclusion bodies, (b) the inclusion bodies are isolated andsolubilized under denaturing conditions, (c) the denatured, monomericand biologically inactive protein, which has been solubilized from theinclusion bodies, is renatured, thereby enabling folding anddimerization to give the soluble, biologically active conformation, and(d) where appropriate, after the renaturation, the mature protein isproteolytically released from its pro form.

[0016] Our in vitro renaturation experiments have shown that it ispossible according to the invention, even in the absence of detergents,to obtain large quantities of biologically active drBMP-2 relativelysimply by using the hypothetical pro form of BMP-2 (Gly²⁰-Arg³⁹⁶) andthen eliminating the propeptide (Gly²⁰-Arg²⁸²). In this connection,drBMP-2 is to be understood as meaning the C-terminal domain of BMP-2from Lys ²⁹⁰, Arg²⁹¹ or Leu²⁹² (see example 2.2.1) to Arg³⁹⁶, thebiological activity of which has been described by Koenig et al., [Mol.Cell Biol. 14 (1994), 5961-5974]. The amino acids (Gln²⁸³ to Arg²⁸⁹,Lys290 or Arg²⁹¹) mediate the binding of BMP-2 to heparin.

[0017] While this interaction, which is, if anything, nonspecific,modulates the effect of BMP-2, it is not essential for the biologicalactivity of the latter [Ruppert et al., Eur. J. Biochem. 237 (1996),295-302].

[0018] According to the invention, either the entire pro sequence(Gly²⁰-Arg²⁸²) or a part of it can be used as the pro sequence orpropeptide.

[0019] In order to increase the rate of expression of probmp-2 inprokaryotes, it is possible, according to the invention, to attach a tagsequence, e.g. a histidine tag, to the 5′ end of the sequence encodingthis gene, or to use mutagenesis to optimize the probmp-2 codons forprokaryotic expression. It is also possible to augment expression asdescribed in U.S. Pat. No. 5,336,602 [Brinkmann et al., 1993] or to usea combination of these methods.

[0020] When recombinant probmp-2 is overexpressed in the cytoplasm ofprokaryotes, the protein then accrues in the cytoplasm in the form ofinactive, insoluble aggregates (inclusion bodies—IBs). In order toisolate them, the cells are first of all disrupted, after fermentation,by means of one or more of the customary methods (e.g.: high pressuredispersion, enzymic lysis or ultrasonication), with this preferablytaking place in a neutral to weak acid buffer solution, for example 0.1M tris/HCl pH 7.0.

[0021] The cellular DNA is degraded, by means of mechanical, chemicalor, preferably, enzymic treatment, into fragments which do notcosediment with the IBs. Insoluble cell constituents, including the IBs,are then separated off from the soluble constituents in any appropriatemanner, with separation by centrifugation being preferred. The pellet iswashed with solutions which, as a result of their composition or ofsubstances added to them, such as detergents, are able to solubilizeinterfering cell proteins and membrane components, but not therecombinant proBMP-2, from the IBs. The remaining, insoluble,proBMP-2-containing fraction is then subsequently solubilized, renaturedand enzymically processed in accordance with the invention.

[0022] Prior to the renaturation, the IBs are solubilized, with usebeing made of customary denaturing agents (chaotropic substances) orcombinations of these agents. These agents include, inter alia,guanidinium salts, e.g. GdmCl or GdmSCN, and also urea and itsderivatives. In this connection, the concentration of the denaturingagent or denaturing mixture is to be adjusted such that the recombinant,difficulty soluble protein can be completely solubilized. In the case ofguanidinium chloride, these concentrations lie in the range of 3-8 Mand, in the case of urea, of 6-10 M. In order to monomerize the soluble,cystine-containing protein, which has thus been denatured, completely aswell, preference is given to adding a reducing agent, such asdithiothreitol (DTT) or β-mercaptoethanol, or a combination of reducingagents, with the pH of the solubilization buffer preferably beingadjusted to between 8 and 9. Following the solubilization, or inparallel with it, it is also possible to covalently modify the freecysteines, for example using oxidized glutathione. After thesolubilization, or the optional covalent modification of the cysteines,the pH of the IB solubilizate is preferably adjusted to 5 or less and,once again optionally, excess redox equivalents are removed by dialysis,with the dialysis buffer, apart from the reducing agent, expedientlyhaving the same composition as the solubilization buffer. Finally, theconstituents which remain insoluble during the solubilization processare separated off (centrifugation or filtration) and discarded. Theoptional modification step, and the removal of the excess reducingagent, enable the IB solubilizate to be prepurified, where appropriate,by means of immobilized metal affinity chromatography (IMAC) providedthe proBMP-2 was expressed with an N-terminal histidine tag.

[0023] The process according to the invention for renaturing rh-proBMP-2involves decreasing the concentration of the denaturing agent(s) down toa level (e.g. ≦0.5 M GdmCl) which does not have a denaturing effect oronly has a weakly denaturing effect. This is preferably effected byslowly diluting the solubilizate, continuously or stepwise, inrenaturation buffer. In order to maximize the yield of biologicallyactive protein, care must be taken to ensure that the intermixing inthis connection takes place as speedily and thoroughly as possible,something which is ensured, for example, by vigorous stirring.

[0024] An alternative to dilution is that of dialyzing the solubilizateagainst renaturation buffer. The conditions are in each case to beselected such that the formation of protein aggregates is minimal.Renaturation of the protein may already take place during the initialdilution of the denaturing agent. After dilution or dialysis, the finalconcentration of rh-proBMP-2 in the renaturation buffer is 10-1 000μg/ml, preferably 50-250 μg/ml.

[0025] Optimal protein concentration is ensured when the unproductiveaggregation of incorrectly folded polypeptide chains and foldingintermediates (a ≧2nd order process [Kiefhaber et al., Biotechnology(N.Y.) 9 (1991), 825-829]) proceeds significantly more slowly than doesthe folding of the individual polypeptide chains to give the nativeconformation (1st order process).

[0026] The pH of the renaturation buffer employed is 6.5-10, ideally8-9. Suitable buffers are consequently any buffers which have a neutralto alkaline pK_(a), preferably tris buffers and phosphate buffers andcombinations of these buffers. It is furthermore expedient to add lowmolecular weight auxiliary agents which increase the renaturation yield[U.S. Pat. No. 5,593,865, Rudolph et al., 1955] to the renaturationbuffer. It is particularly suitable to renature in the presence ofL-arginine, with use being made of concentrations of 0.2-2.0 M,preferably 0.5-1.5 M, L-arginine. Another component of the renaturationbuffer is, according to the invention, a combination of the reduced andoxidized forms of one or more thiol compounds. Examples of suchcombinations are reduced (GSH) and oxidized (GSSG) glutathione, cysteineand cystine, cysteamine and cystamine, and also β-mercaptoethanol and2,2′-hydroxyethyl disulfide. An artificial redox system of this naturecan be used to control the renaturation of the proteinthermodynamically. The possibility of forming the disulfide bridges,which is necessary for the correct folding of the polypeptide chains[Creighton, Biochemistry 378 (1997), 731-744], is ensured by thepresence of the oxidized form, while the possibility of rebreakingincorrectly formed cystines (reshuffling) is ensured by the presence ofthe reduced form of the thiol component [Creighton et al., TrendsBiotechnol. 13 (1995), 18-23].

[0027] Preference is given to using glutathione in reduced (GSH) andoxidized (GSSG) form at concentrations of in each case up to 10 mM,ideally in a ratio of 5 mM GSSG to 2 mM GSH.

[0028] According to the invention, the renaturation buffer preferablycontains one or more chelating agents, such as EDTA(ethylenediaminetetraacetic acid), at concentrations of up to 20 mM(preferably approx. 5 mM) in order, on the one hand, to prevent theuncontrolled, accelerated oxidation of the reduced thiol component bymetal ions and, on the other hand, to inhibit metalloproteases whichmight contaminate the renaturate.

[0029] According to the invention, the renaturation is expedientlyeffected at temperatures of 0-20° C. over 1-21 d, preferably 5-10° C.and 5-10 d.

[0030] The relatively long renaturation times can be explained by thecovalent dimerization of the proBMP-2 polypeptide chains. Ourexperiments have shown that, under the conditions of the presentinvention, it is the oxidation of free thiol groups, and not the proteinconcentration, which determines the rate of this process.

[0031] After the renaturation process has been concluded, therenaturation mixture is, according to the invention, dialyzed against apreferably acid (pH≦5) buffer which does not contain any low molecularweight auxiliary agents within the meaning of the present invention. Inthis connection, the dialysis buffer conforms to the process accordingto the invention when the dialysis, to the greatest possible extent,aggregates incorrectly folded polypeptide chains and foldingintermediates, which can consequently be separated off by means ofcentrifugation, filtration or another customary method. Prior to thedialysis, it is expedient to concentrate the renaturation mixture, forexample by means of cross-flow filtration, which can be followed by anadditional incubation time under renaturation conditions. Aerating thebuffer, something which may also possibly take place in association withthe process of concentrating, and which accelerates the oxidation offree thiol groups, may have the effect of shortening the renaturationtime. This can also be achieved by adding substances which have anoxidizing effect.

[0032] According to the invention, chromatographic separating methodscan be used to separate monomeric, incorrectly folded or non-dimerized,correctly folded species from the native, dimeric form of the renaturedrh-proBMP-2, with preference being given to using reversed-phase HPLC.Suitable column materials for this purpose, are, in particular,silica-based C4-matrices or polymer-based particles of comparable orstronger hydrophobicity, with a pore diameter of ≧300 Å being advisabledue to the size of the protein. Suitable eluents are those which arecustomary in RP-HPLC, such as acetonitrile, 2-propanol or ethanol.According to the invention, use is made, in this connection, of ion-pairformers, preferably trifluoroacetic acid or perfluorobutyric acid, atthe customary concentrations.

[0033] Another possibility for purifying dimeric, naively foldedproBMP-2 is that of using heparin affinity chromatography. In thisconnection, urea concentrations of from 2 to 8 mol/l, preferably 5mol/l, should be used as an addition to the buffer in order to increasethe solubility of the protein and avoid nonspecific interactions betweenproBMP-2 and the column material. HiTrap Heparin-Sepharose (AmershamPharmacia Biotech) is a particularly suitable column material in thiscontext. The monomeric forms of proBMP-2 are then eluted from the matrixat less than 300 mol of NaCl/l while the dimeric form is eluted athigher salt concentrations.

[0034] WO 00/22119 [Rattenholl et al., 2000] has already described amethod for isolating the biologically active protein from its inactive,denatured pro form in the case of the nerve growth factor β-NGF, whichdoes not belong to the TGF-β family. β-NGF and BMP-2 differ, inparticular, in the length of their pro sequence (β-NGF-103 AA; BMP-2-263AA). There are also significant differences in the tertiary structure[McDonald and Hendrickson, Cell 73 (1993), 421-424]. It is notsurprising, therefore, that the present invention differs from WO00/22119 in essential points.

[0035] The renaturation times which are required for maximal yields ofcorrectly folded rh-proBMP-2 are significantly longer (1-21 d) than arethose for rh-proNGF (3 h). In vivo, the pro form of BMP-2, like that ofβ-NGF, is processed by prohormone convertases of the furin type. Theseconvertases recognize a dibasic sequence motif at the end of the prosequence. Their last amino acid is Arg²⁸². In vitro, proteases havingcomparable substrate specificity are able to eliminate the BMP-2propeptide or β-NGF propeptide.

[0036] However, the processing of the renatured rh-proBMP-2 requiresentirely different conditions from those employed for cleaving proNGF.Although the BMP-2 pro sequence which is used in accordance with theinvention is, with 263 AA, substantially longer than the mature protein(114 AA), it has surprisingly been found, in our experiments, that thesolubility properties of proBMP-2 are determined by its mature domain.In contrast to the BMP-2 propeptide on its own (Gly ²⁰-Arg²⁸²), proBMP-2is therefore only soluble at high concentrations, without anysolubilizing additives, at a pH of ≦5. At acid pH, it is not possible touse proteases (trypsin, PACE (paired basic amino acid-convertingenzymes; eukaryotic endopeptidases which cut after dibasic motifs)) toefficiently process the pro form. However, in order to enableproteolytic cleavage to take place, use is made, according to theinvention, of any noninhibiting solubilizers, in particular urea ortrishydroxymethylaminomethane (tris) at concentrations of 1-5 M and0.1-2 M, respectively, either individually or in combination, preferably2-4 M urea in combination with 0.1-1 M tris. Using these additives, itis possible to keep proBMP-2 in solution at concentrations of ≧1 mg/mleven at the pH optimum (7-8) of the proteases employed.

[0037] Dialysis is expediently used to adjust the buffering conditionsfor the proteolysis. According to the invention, preference is given totrypsin-like proteases, which are used in a mass ratio of from 1:10 to1:1 000 (trypsin, rh-proBMP-2), preferably of from 1:20 to 1:100. Themixture is incubated over a period of from 1 min to 24 h, preferably offrom 30 min to 6 h, at a temperature of 0-37° C., preferably 10-25° C.In the case of tris, the solubilizer itself functions as the bufferingsubstance while, in the case of urea, tris buffer, which is added inaddition at the given concentrations, functions as the bufferingsubstance. Furthermore, a high concentration of tris, e.g. 0.5 M,prevents the covalent modification of amino acid side chains byisocyanate which may have been formed from the urea.

[0038] The pH is adjusted to a value which is favorable for the proteaseconcerned, preferably 7-8. In order to suppress nonspecific proteolyticdegradation by metalloproteases, it is expedient, according to theinvention, to add chelating agents, e.g. 1-20 mM EDTA. The proteolysisis stopped by adding one or more protease inhibitors, preferably 1-5 mMphenylmethylsulfonyl fluoride (PMSF) or trypsin inhibitor obtained frombovine pancreas or soybeans (BPTI/SBTI) in a 2-20-fold molar excessrelative to the trypsin, or by using acid to adjust the pH to ≦5[Rudolph et al.; Folding proteins, in Protein Function: A PracticalApproach, T. E. Creighton (Ed.), 2nd Edtn. (1997), 57-99].

[0039] The N-terminus of the mature BMP-2 (Gln²⁸³-Arg²⁹¹) isunstructured and therefore protease-sensitive. In addition, it containssome arginine and lysine residues (Arg^(289, 291), LyS^(285, 287, 290))The limited proteolysis with trypsin, which preferably cuts after thesebasic amino acid residues, therefore also leads to the flexibleN-terminus of mature BMP-2 being eliminated. The biological activity ofthe resulting drBMP-2 has been demonstrated in animal experiments.According to the invention, it is possible, where appropriate by meansof protein engineering, to introduce an additional disulfide bridge forthe purpose of fixing and stabilizing the mature BMP-2 N-terminus, e.g.as is present in the related TGF-β, in order, in this way, to protectthe BMP-2 heparin-binding site from proteolytic degradation. Ourexperiments on the limited proteolysis of rh-proBMP-2 in the absence ofsolubilizer concentrations according to the invention, at proteinconcentrations which are then correspondingly low, have shown that thepropeptide consists of at least one domain which is still largelyprotease-resistant following elimination from the mature protein. Someof the solubilizers which are used themselves have a weakly denaturingeffect at the concentrations employed. This results in the BMP-2propeptide, which is not stabilized by intracatenary disulfide bridges,being structurally destabilized and protease-labile and consequentlybeing able to be degraded under the conditions of limited proteolysisaccording to the invention.

[0040] Because of the extreme hydrophobicity of native BMP-2 [Scheufleret al., J. Mol. Biol. 287 (1999), 103-115], purification by way ofhydrophobic interactions suggests itself as a method for purifying theproteolysis products. According to the invention, preference is given tousing, for this purpose, column materials to which the drBMP-2 which hasbeen obtained binds strongly, whereas the other degradation products,and the protease employed and/or the inhibitor, only interact weaklywith the material. In conformity with the present invention, it ispossible to alter the buffering conditions after the proteolysis suchthat the drBMP-2 precipitates while the other degradation products, theprotease and/or its inhibitor(s) remain in solution. After it has beenseparated from the supernatant, the precipitated drBMP-2 can then besolubilized once again in suitable buffers or solvents. This change toprecipitating conditions in the presence of a suitable column material,in particular “Fractogel EMD Phenyl S” (Merck) or comparable materials,such as Phenylsepharose (Pharmacia), is preferably effected by means ofdialysis. If the material is used in a large excess, it is possible toachieve the situation where the protein binds to this material before itaggregates. After the column material has been loaded with protein inthis way, the unwanted components can be removed by means of suitablewashing steps. The drBMP-2 is expediently eluted under conditions whichare mildly to strongly denaturing but in no way reducing. Depending onthe column material, suitable eluents are accordingly, inter alia,arginine, urea or guanidinium salts which, in low concentrations, canalso be used for washing the column material. After the elution, thedrBMP-2-containing fractions are, where appropriate, combined and,particularly preferably, dialyzed against 0.1% trifluoroacetic acid(TFA) or ammonium acetate, which are very suitable solvents for thefinal application of BMP-2. According to the invention, it is alsopossible to separate the proteolysis products using reversed-phasematerials, preferably C₄-RP-HPLC, with the elution preferably beingcarried out using 0.1% TFA in an acetonitrile gradient.

[0041] Mature BMP-2, which is renatured in accordance with the inventionand obtained after eliminating the pro sequence, can be used, in themanner which is customary for this purpose, for promoting bone growth.Surprisingly, however, the renatured proBMP-2 itself, which stillcontains the entire pro sequence or a truncated pro sequence, is alsoequivalent to the mature BMP-2 in its biological activity and can beused like the mature BMP-2. Part of the subject matter of the inventionis therefore also a pharmaceutical for promoting bone growth whichcomprises proBMP-2 as the active compound. The proBMP-2 can contain thecomplete pro sequence or a truncated pro sequence. Preference is given,according to the invention, to using a combination of proBMP-2 andvarious medical implant materials for both temporary bone replacement(bone replacement materials) and permanent bone and joint replacement(metal implants such as hip and knee joint endoprostheses) intraumatology, orthopedics, ear, nose and throat surgery, oral andmaxillofacial surgery and neurosurgery. In this connection, proBMP-2can, in the first place, be used as an additional implant coating in thenature of a thin protein film which adheres, with or without adhesives(e.g. silane), to all the inner and outer surfaces. In the second place,proBMP-2 can be used as an integral constituent of implants by adding itto the nonsolid precursors of those solid end products which can beprepared by chemical polymerization or curing (e.g. calcium phosphatecements such as Norian SRS, Ostim, and others).

[0042] Inorganic and organic materials of natural or artificial origin,and also mixed forms of these two materials, are suitable for use asimplants for transient bone replacement. Examples of inorganic bonereplacement materials are ceramic materials composed of hydroxyapatite,beta-tricalcium phosphate, alpha-tricalcium phosphate, glass ceramicmaterials, bioglasses, calcium carbonates, calcium sulfates and other(not listed) calcium salts in crystalline and amorphous form, includinginjectable calcium phosphate-based bone cements. Suitable organicmaterials are, inter alia, collagens, bone tissue, demineralized bonematrix, gellable polysaccharides such as hyaluronic acid, biocompatiblesynthetic materials, such as polylactic acid, polymethyl acrylates,polyethylene glycols, and others, in the widest possible variety ofmixed forms and processing forms. Endoprostheses and other metalimplants, such as those for vertebral stabilization, are suitable forpermanent bone replacement. These latter are customarily productscomposed of different stainless steels and titanium alloys, some ofwhich are also provided with hydroxyapatite coatings.

[0043] Other parts of the subject matter of the invention are thereforeproBMP-2 possessing the complete or truncated pro sequence to which themature BMP-2 is bonded, a pharmaceutical comprising this substance and aprocess for producing a pharmaceutical for promoting bone growth asdefined in the patent claims. The complete sequence of proBMP-2 fromGly²⁰-Arg³⁹⁶, including 20 amino acids of His tag and the start codonMet, is shown in table 2. The amino acids Met1 to His20 are aconstituent of the pET-15b-encoded His tag. The pro sequence of BMP-2begins at Gly²². The additional methionine is located between the Histag and the pro sequence. Gly²² to Arg³⁹⁸ in the sequence depicted herecorrespond to Gly²⁰ to Arg³⁹⁶ in the primary translation product of thehuman preproBMP-2 cDNA, to which the sequence data in the text refer.

[0044] The following examples explain the invention in combination withthe drawing. In this drawing:

[0045]FIG. 1 depicts an SDS-PAGE electropherogram

[0046]FIG. 2 depicts UV-CD spectra

[0047]FIG. 3 depicts emission spectra, in each case of proBMP-2

[0048]FIG. 4 depicts an SDS-PAGE electropherogram

[0049]FIG. 5 depicts an RP-HPLC-chromatogram

[0050]FIG. 6 depicts UV-CD spectra

[0051]FIG. 7 depicts a MALDI-TOF mass spectrogram, in each case ofdrBMP-2

[0052]FIG. 8 depicts an RP-HPLC diagram of rh-proBMP-2

[0053]FIG. 9 depicts a histological section of an rh-proBMP-2 implant.Both the inner and the outer surface of the ceramic are coated with bonesubstance. The interior of the pore contains bone marrow containingnumerous fat cells (implant material—dark, bone—light blue). Some musclefibers (dark blue) and connective tissue can be seen at the bottom left.

[0054]FIG. 10 depicts a histological section of an rh-proBMP-2 implantat high magnification. The bone material (top left to bottom right,light blue) is in very close contact with the ceramic material which itcovers (on the right, grey). Active osteoblasts are present in a rowbetween bone substance and hematopoietic tissue.

[0055]FIG. 11 depicts a comparison of the activity of AP in theimplants.

EXAMPLES Implementation Example 1 Preparing proBMP-2

[0056] Cloning the Probmp-2 cDNA into an Escherichia coliExpressionVector

[0057] The T7 expression system supplied by Novagen [Studier and Moffat,J. Mol. Biol. 189 (1996), 113-130] was used for cloning the probmp-2cDNA.

[0058] The pcDNA3 vector (Boehringer-Mannheim GmbH) containing insertedpreprobmp-2 cDNA was used as the starting DNA.

[0059] The DNA encoding amino acids Gly²⁰ to Arg³⁹⁶ of BMP-2 wasobtained by polymerase chain reaction (PCR). In this reaction, use wasof mutagenesis primers to insert a methionine codon, for the translationstart, at the 5′ end, to convert the stop codon at the 3′ end into an E.coli-typical stop codon and to supplement it with a second stop codon.In addition, the primers respectively contained an NdeI cleavage site(5′ end) and a BamHI cleavage site (3′ end). This enabled the subsequentcloning into the expression vector pET-15b (Novagen) to take place. Thisvector additionally encodes an N-terminal 6-histidine tag which enabledthe expression rates in E. coli to be substantially increased.

[0060] After the conclusion of the cloning, the nucleotide sequence waschecked by DNA sequencing [Sanger et al., Proc. Natl. Acad. Sci. USA 74(1977), 5463-5467].

[0061] The following primers were used: forward primer “PrimerFwProBMP”:                NdeI 5′-cg gaa ttc   ca↓t atg ggt gcg gct ggc ctc gttcc-3′                 Met Gly Ala Ala Gly Leu Val                 H₂N →reverse primer “PrimerRevBMP”:        BamHI 5′-cc  g↓ga tcc tta cta gcgaca ccc aca acc-3′                 Stp Stp Arg Cys Gly Cys Gly                HOOC←

[0062]E. coli BL21 (DE3) was used as the host strain. The chromosome inthese bacteria contains the gene for T7-RNA-Polymerase, which is underthe control of the lac promoter. Expression of the polymerase cantherefore be induced with isopropyl-β-D-thiogalactoside (IPTG). Theprobmp-2 gene is controlled by a T7 promoter. Induction with IPTGconsequently leads to the formation of proBMP-2. In order to furtherincrease the expression rate, the cells were cotransformed with pUBS520.This plasmid contains the gene for an arginine tRNA (dnay) which is rarein E. coli. On the other hand, the arginine codons (AGA and AGG) whichthis tRNA recognizes occur particularly frequently in eukaryotic genesand therefore also in probmp-2. The expression rate of these genes in E.coli depends, inter alia, on the availability of this tRNA [Brinkmann etal., Gene 85 (1989), 109-114].

[0063] Expressing Human ProBMP-2 in E. coli

[0064] In order to propagate the recombinant bacterial strain, 100 μg ofampicillin/ml and 50 μg of kanamycin/ml were added to a suitable volume,as a rule 1.5 l, of 2·YT medium, which was inoculated with a singlecolony.

[0065] 2·YT Medium (1 l):

[0066] 17 g of tryptone

[0067] 10 g of yeast extract

[0068] 5 g of NaCl

[0069] The culture was shaken at 160-200 rpm and 37° C. The culture wasinduced with 1 mM IPTG at an OD₆₀₀ of 1-1.3. The culture was then shakenat the same temperature for a further 3-4 h and, in this way,reproducible cell densities following induction of OD₆₀₀ 3-3.5 wereachieved. The cells were harvested by centrifuging at 10 000 g. In thisway, it was possible to obtain 3-3.5 g of cells (wet weight) per 1 l ofmedium. As an alternative to immediate cell disruption, the cells werefirst of all frozen in liquid nitrogen and, after that, at −20° C.

[0070] Isolating the Inclusion Bodies (IBs)

[0071] When recombinant probmp-2 is expressed, aggregates, i.e.inclusion bodies (IBs), form in the bacteria cells. These IBs wereprepared as described in Rudolph et al.: “Folding proteins”, in “ProteinFunction: A Practical Approach”, T. E. Creighton (Ed.), 2nd Edtn.(1997), 57-99.

[0072] In each case 5 g of cell pellet were resuspended in 25 ml of 100mM tris/HCl pH 7.0, 1 mM EDTA. After 1.5 mg of lysozyme had been addedper 1 g of wet cell weight, the cell suspension was then incubated for30 minutes at 4° C. Finally, the cells were disrupted by means of highpressure dispersion (Gaulin homogenizer). After 3 mM MgCl₂ and 10 μg ofDNase/ml had been added, the homogenate was incubated for a further 30min at 25° C. 0.5 part by volume of 60 mM EDTA, 6% Triton X-100, 1.5 MNaCl, pH 7.0, were then added and the incubation was continued at 4° C.for 30 min in order to solubilize insoluble cell constituents. Underthese conditions, the IBs remained insoluble and could be separated offby centrifuging at 39 000 g. In order to remove excess detergent fromthe IBs again, the latter were washed a further 5 times with 100 mMtris/HCl, pH 7.0, 20 mM EDTA and finally aliquoted and stored at −20° C.

[0073] In this way, it was possible to reproducibly obtain 180-210 mg ofIBs, which, at a protein content of 30-35%, contained 90-95%rh-pro-BMP-2, from 1 g of induced E. coli cells.

[0074] Solubilizing the IBs

[0075] 100-150 mg of IB pellet were solubilized, at 25° C. for 4 h, in 1ml of 100 mM tris/HCl, pH 8.0, 6 M GdmCl, 100 mM DTT, 1 mM EDTA. Afterthat, the pH was adjusted to 3-4 with acetic acid and the insolubleconstituents were pelleted at 16 000 g for 20 min in a centrifuge. Thesupernatant was then centrifuged once again for 30 min in anultracentrifuge at 266 000 g in order to separate off microaggregates.The protein concentration was determined by measuring absorption atλ=280 nm [Gill and von Hippel, Anal. Biochem. 182 (1989), 319-326] andwas 30-52 mg/ml.

[0076] Purifying ProBMP-2

[0077] Recovery rate (approx. 70%) in this connection was comparablewith the analytical scale.

[0078] Heparin affinity chromatography on a 1 ml HiTrap HeparinSepharose (Amersham Pharmacia Biotech) was used as a second purificationmethod. The buffer system employed was:

[0079] 0.1 mol 1⁻¹ tris, pH 7.56, 6 mol 1⁻¹ urea (buffer A), and

[0080] 0.1 mol ¹⁻¹ tris/HCl, pH 7.5, 6 mol 1⁻¹ urea, 1 mol ⁻¹ NaCl(buffer B).

[0081] The column was equilibrated with more than 10 column volumes ofbuffer A. Impurities were eluted using a gradient of from 0 to 20%buffer B. Monomeric and dimeric proBMP-2 were separated using a gradientof from 20 to 50% buffer B in 30 min at a flow rate of 1 ml min⁻¹.

[0082] Renaturing Rh-proBMP-2 on a Preparative Scale

[0083] The proBMP-2 IB solubilizate, containing 4 M GdmCl, pH 4, wasdiluted for the renaturation such that, after complete dilution of thesolubilizate in folding buffer, the final concentration of GdmCl reached50-60 mM. The final concentration of proBMP-2 was 3-4 μm (corresponds to135-180 μg/ml).

[0084] Folding Buffer:

[0085] 100 mM tris/HCl, pH 8.0

[0086] 1 M L-arginine

[0087] 5 mM GSSG

[0088] 2 mM GSH

[0089] 5 mM EDTA

[0090] After the folding buffer had been filtered and degassed, thesolubilizate was slowly added (˜20 ml/h), while stirring, and themixture was incubated at 5° C. for 7 d. In this connection, the. DTTresidues which were entrained with the solubilizate are equivalent to aconcentration of at most 0.4 mM GSH.

[0091] The folding mixture was concentrated by means of cross-flowfiltration (Filtron Minisette, membrane: 30 kDa open channel). Theconcentrate was then dialyzed five times against 20 volumes of 50 mM Naacetate, pH 5.0, for in each case 24 h and, after that, aggregatedprotein was centrifuged off at 75 000 g for 2 h. The quantity of proteinremaining in solution was determined by absorption at λλ32 280 nm andwas reproducibly approx. 50% based on the protein content of theinitially employed IB solubilizate.

[0092] Purifying ProBMP-2

[0093] RP-HPLC was employed for the analytical or semipreparativeseparation of the (incorrectly or correctly folded) monomeric speciesfrom native, dimeric rh-proBMP-2. On the analytical scale, use was made,inter alia, of a protein RP column (150×3.6 mm, YMC) using 0.1%perfluorobutyric acid (HFBA) in H₂O or ACN as the mobile phase (see FIG.8). Separation of monomer and dimer was also achieved usingtrifluoroacetic acid as ion-pair former. Because of its higherhydrophobicity, the dimer, as in the case of mature BMP-2, elutes at alater stage than does the monomer or the monomeric, incorrectly foldedspecies. On the semipreparative scale, use was made of a columncontaining Source 15 RPC material (Pharmacia). In this connection, theseparation profile and the recovery rate (approx. 70%) were comparablewith the analytical scale.

[0094] Characterizing Rh-proBMP-2

[0095] Purity Analysis and Molecular Weight Determination by Means ofSDS-Polyacrylamide Gel Electrophoresis

[0096] The SDS-PAGE analysis indicated that the proBMP-2 IBs which hadbeen prepared in accordance with the invention were highly pure (≧90%;FIG. 1, lanes 2-5), which meant that it was possible to use them for therenaturation without any prior purification.

[0097] SDS-PAGE carried out under nonreducing conditions (addition ofiodoacetamide leads to irreversible, oxidative modification of freethiol groups) shows that renatured proBMP-2 exhibits a high degree ofcovalent dimerization (≧90%), with this indicating that the disulfidebridges have been correctly formed and that the protein possesses thenative structure (FIG. 1, lane 6).

[0098] N-Terminal Sequence Analysis

[0099] The N-terminal sequencing [Edman and Begg, Eur. J. Biochem. 1(1967), 80-91] was carried out after performing SDS-PAGE and Westernblotting onto a PVDF membrane. In the case of renatured proBMP-2, thesequencing gave the following amino acid sequence: Gly-Ser-Ser-His-His-

[0100] This corresponds to the N-terminus of recombinant proBMP-2possessing a histidine tag, after the starting methionine has beeneliminated.

[0101] Circular Dichroism Spectrometry and Fluorescence Spectroscopy

[0102] The fluorescence-spectrometric and CD-spectrometric analyseswhich were carried out on renatured proBMP-2 and on proBMP-2 which hadonce again been denatured provide clear indications of the presence ofsecondary and tertiary structure. The marked difference between theellipticity of renatured and denatured proBMP-2 in the region aroundλ=220 nm of the far UV CD spectrum (FIG. 2) makes it possible toconclude that secondary structure elements are formed in therenaturation process according to the invention. The shifting of theemission maximum of the fluorescence of renatured proBMP-2 toward lowerwavelengths as compared with the denatured protein (FIG. 3) is typicalfor the formation of native tertiary structures. In this connection, thetryptophan residues of the protein (5 tryptophans per proBMP-2polypeptide chain) pass from the polar environment of the solvent intothe apolar core of the folding protein, where they are also stericallyfixed. The excitation energy loss which has been reduced in this wayresults in light of higher frequency being emitted. [Schmid: “Opticalspectroscopy to characterize protein conformation and conformationalchanges.” in “Protein Structure: A Practical Approach”, T. E. Creighton(Ed.), 2nd Edtn. (1996), pp. 261-297]. It is also clear from thefluorescence spectra that the structure of proBMP-2 undergoes a changeat low pH, that is in the region where mature BMP-2 and proBMP-2 arehighly soluble. As our experiments have indicated, this may beattributed to the propeptide unfolding in part (data not shown).

[0103] MALDI-TOF Mass Spectrometry and N-terminal Sequence Analysis

[0104] apparent molecular mass: 89803.0078 Da—see FIG. 7

[0105] calculated molecular mass (without the starting methionine):89801.2 Da

[0106] Biological Activity of Rh-proBMP-2 and Rh-drBMP-2

[0107] The osteoinductive activity of the modified rh-proBMP-2 andrh-drBMP-2 proteins was examined in an ectopic bone induction modelusing small laboratory animals. For this, defined quantities of thegiven factor to be tested (40-200 μg; drBMP-2 or proBMP-2) were applied,after having been sterilized by filtration, to sterile cubiform,chemically and pharmacologically inert ceramic implant bodies of β-TCP.Implant specifications: Manufacturer Mathys, Bettlach, Switzerland Tradename chronOs Phase purity >95% β-TCP Pore volume fraction 72 +/− 6% Poresize 80-650 μm Pore structure interconnecting Solidity 7.7 +/− 1.2 MPa

[0108] The factor-coated implants, and also uncoated but otherwiseidentically treated control implants, were implanted in the anteriorabdominal wall musculature of 8 fully grown laboratory rats. For this,several approx. 1 cm-long surgical incisions were made in the skin, andthe uppermost muscle layer lying below it, after the rats had beenanesthetized. Blunt preparation was used to produce a cavity between thetwo oblique layers of abdominal musculature, after which the implantswere introduced into this cavity and the wounds were closed withsurgical sutures. After the implants had been left in place for 30 days,the animals were sacrificed and the implants, together with thesurrounding tissue, were removed and examined macroscopically andhistologically for bone neoformation. In the case of all thefactor-coated implants, it was possible histologically to clearlyobserve bone neoformation accompanied by the formation ofhematopoietically active bone marrow (see FIGS. 8 and 9). Consequently,the biological activity was 100% in the case of both factors (16/16 inthe case of proBMP-2; 8/8 in the case of drBMP-2). It was consequentlypossible to unambiguously provide evidence of osteoinductive activity bythe formation of bone tissue in muscle tissue, which is not normallycapable of forming bone. It was not possible to observe any qualitativedifferences in the histological result between proBMP-2 and maturedrBMP-2. In no case did the control implants lead to any boneneoformation. Instead of this, they were only enclosed in connectivetissue.

[0109] Further evidence of the neoformation of active bone tissue underthe influence of rh-proBMP-2 or rh-drBMP-2 was furnished by measuringthe activity of alkaline phosphatase (AP) in the implants. To do this,half of each implant was deep-frozen immediately after explantation andstored at −80° C. For extracting the alkaline phosphatase anddetermining its enzymic activity, the implants were thawed and disruptedin 0.1 M tris/HCl, 1% Triton X-100, pH 7.4 (at 4° C.) using a Potter Shomogenizer (B. Braun Biotech), and insoluble constituents wereseparated off by centrifugation. The clear supernatant was then used fordetermining the alkaline phosphatase activity using the Ecoline 25(Merck) test kit. The concentration of the 4-nitrophenol which wasliberated in this test was monitored for 5 min at 25° C. and λ=405 nmand the enzymic activity was calculated using equation 1 $\begin{matrix}{{{Act}.}:\quad \frac{{Vb} + {{{Vs} \cdot \Delta}\quad {E \cdot 1000}}}{{ɛ \cdot {Vs} \cdot d}\quad t}} & (1)\end{matrix}$

[0110] where Vb is the volume of the buffer (1 ml) and Vs is that of thesample (25 ml). In conformity with the manufacturer's instructions, ε=18518 l×mol⁻¹×cm⁻¹ was assumed to be the molar absorption coefficient of4-nitrophenol. The units of the resulting activity areμmol×min^(×1)×ml⁻¹. The results of the test are summarized in Table 1and FIG. 10. While the enzymic activity in the rh-pro/drBMP-2-containingsamples is significantly increased as compared with the (−) controls,that of the rh-drBMP-2 samples is only slightly increased as comparedwith that of the samples containing rh-proBMP-2 and not in every case,either (exceptions: animals 4, 6 and 8). Taking into consideration thedifferent molecular weights of rh-proBMP-2 (approx. 45 kDa) andrh-drBMP-2 (approx. 12 kDA) and also the fact that the rh-proBMP-2samples were not used in purified form, and consequently still containedincorrectly folded, monomeric species in a proportion of up to 10%, itcan be concluded that, in this animal model, rh-proBMP-2 possesses abone neoformation-inducing activity which is comparable to that of the(truncated) mature form of the protein. TABLE 1 Comparison of thealkaline phosphatase activity (in U) in the β-TCP implants coated withdrBMP-2 or proBMP-2 Animal Control drBMP-2 proBMP-2(a) proBMP-2(b) 10.0009 2.1162 1.1654 1.049 2 0.0232 1.3459 0.7443 0.9126 3 0.017 2.53071.3521 1.4508 4 0.0036 1.0461 1.1934 1.262 5 0.0118 1.3884 1.0472 0.68956 0.0142 0.9195 1.7609 1.257 7 0.0112 1.776 1.4601 1.1126 8 0.01490.7044 1.6968 1.053

Implementation Example 2 Obtaining Biologically Active drBMP-2

[0111] Limited Proteolysis of Rh-proBMP-2

[0112] For the preparative proteolysis of rh-proBMP-2, 50 ml of therenatured protein were dialyzed against 0.1 M tris/HCl pH 7.0, 4 M urea,1 mM EDTA. Aggregates were separated off by centrifugation (75 000 g, 1h) and the protein concentration was determined spectrophotometrically(1.3 mg/ml). The losses due to the dialysis amounted to 13%. Trypsin(Roche Diagnostics) was used in a ratio of 1:100 (Trypsin:proBMP-2;w/w), and the mixture was incubated at 5-7° C. for 4 h. The trypsin wasinactivated with an 8-fold molar excess of soybean trypsin inhibitor(SBTI; Sigma). The entire mixture was dialyzed against urea-free buffer(0.1 M tris/HCl, pH 7.0) in the presence of hydrophobic column material(20 ml of Fractogel-Phenyl, Merck). The column material, which had beenloaded with drBMP-2 in this way, was packed into an empty column (XK-16,Pharmacia) and then washed with:

[0113] 5 CV (column volumes) of low salt buffer (dialysis buffer),

[0114] 5 CV of 50 mM Na acetate, pH 5.0,

[0115] 2 CV of 50 mM Na acetate, pH 5.0, 4 M urea, and

[0116] 2 CV of 50 mM Na acetate, pH 5.0, 1 M L-arginine.

[0117] The column was eluted with 6 M GdmCl, 0.2 M acetic acid. ThedrBMP-2-containing fractions were combined and dialyzed against 0.1%TFA. It was not possible to observe any aggregate formation. Thedialyzate was sterilized by filtration and the protein concentration wasdetermined spectrophotometrically (1.3 mg/ml). The yield of BMP-2 whichwas obtained in this way was approx. 50% based on the renaturate whichwas used for the limited proteolysis. Consequently, approx. 17 mg ofbiologically active drBMP-2s can be obtained from 1 l of E. coli cultureor 5.2 (corresponds to 5.6 mg of mature BMP-2 incl. N-terminalheparin-binding site) based on 1 g (wet weight) of induced E. colicells. By contrast, only 0.2 mg of BMP-2 was obtained per 1 g of cellsusing the method described by Ruppert et al., [Eur. J. Biochem. 237(1996), 295-302].

[0118] Characterizing drBMP-2

[0119] Analyzing the Proteolysis Products by Means of N-terminalSequencing

[0120] The N-terminal sequencing [Edman and Begg, Eur. J. Biochem. 1(1967), 80-91] was carried out after performing SDS-PAGE and Westernblotting onto a PVDF membrane. The sequencing gave the following aminoacid sequences for drBMP-2 which was generated in accordance with theinvention: a) Leu-Lys-Ser-Ser-Cys-Lys-Arg- b)Lys-Arg-Leu-Lys-Ser-Ser-Cys- c) Arg-Leu-Lys-Ser-Ser-Cys-Lys-

[0121] The ratio of the different species to each other is about 3:2:1(a:b:c). While it was not possible to detect the cysteines, it waspossible to detect the amino acids which followed them. However, thesequence was completely verified at the DNA level. It corresponds to theN-terminus of mature BMP-2 without the heparin binding site. drBMP-2 wasused for the sequencing both in the reduced form and in the nonreducedform. It was only possible to sequence the nonreduced form up to thesecond serine, suggesting that the following residue is a cystine, thatis a constitutent of a disulfide bridge. Taken overall, the proteolysisproduct consequently corresponds to a mature BMP-2 which has beentruncated N-terminally by 7-9 amino acids and which therefore lacks theheparin binding site.

[0122] Under the limited proteolysis conditions described here, peptidechains which are not disulfide-bridged are degraded, meaning that, inthe end, it is only drBMP-2 which is 100% covalently dimerized which isobtained.

[0123] Analyzing drBMP-2 for Purity Using SDS-Polyacrylamide GelElectrophoresis and HPLC

[0124] The size and purity of the drBMP-2 which was obtained inaccordance with the invention was checked by carrying out SDS-PAGE underreducing and nonreducing conditions and then carrying out silverstaining (lanes 6 and 11 in FIG. 4). It is 100% dimerized and is morethan 95% pure.

[0125] In addition, reversed-phase high performance liquidchromatography (RP-HPLC; Vydac C₄) was used for checking the purity andhomogeneity of drBMP-2. In this experiment, drBMP-2 eluted in ahomogeneous peak at 46.57% acetonitrile and 0.1% TFA (FIG. 5), providingsupport for the uniform structuring and, in particular,disulfide-bridging, of the protein.

[0126] Circular Dichroism Spectrometry

[0127] The marked difference between the ellipticity of renatured anddenatured drBMP-2 and, in particular, of reduced and denatured matureBMP-2 (IB solubilizate) in the 210-230 nm region of the far UV CDspectrum (FIG. 6) makes it possible to conclude that, in the processaccording to the invention for renaturing the proprotein, secondarystructure elements are formed in the mature domain of the protein aswell. TABLE 2 Subject: 3 letter code proBMP (incl. 20 AA His tag)MetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerHisMetGlyAlaAlaGlyLeuValProGluLeuGlyArgArgLysPheAlaAlaAlaSerSerGlyArgProSerSerGlnProSerAspGluValLeuSerGluPheGluLeuArgLeuLeuSerMetPheGlyLeuLysGlnArgProThrProSerArgAspAlaValValProProTyrMetLeuAspLeuTyrArgArgHisSerGlyGlnProGlySerProAlaProAspHisArgLeuGluArgAlaAlaSerArgAlaAsnThrValArgSerPheHisHisGluGluSerLeuGluGluLeuProGluThrSerGlyLysThrThrArgArgPhePhePheAsnLeuSerSerIleProThrGluGluPheIleThrSerAlaGluLeuGlnValPheArgGluGlnMetGlnAspAlaLeuGlyAsnAsnSerSerPheHisHisArgIleAsnIleTyrGluIleIleLysProAlaThrAlaAsnSerLysPheProValThrArgLeuLeuAspThrArgLeuValAsnGluAsnAlaSerArgTrpGluSerPheAspValThrProAlaValMetArgTrpThrAlaGluGlyHisAlaAsnHisGlyPheValValGluValAlaHisLeuGluGluLysGlnGlyValSerLysArgHisValArgIleSerArgSerLeuHisGlnAspGluHisSerTrpSerGlnIleArgProLeuLeuValThrPheGlyHisAspGlyLysGlyHisProLeuHisLysArgGluLysArgGlnAlaLysHisLysGlnArgLysArgLeuLysSerSerCysLysArgHisProLeuTyrValAspPheSerAspValGlyTrpAsnAspTrpIleValAlaProProGlyTyrHisAlaPheTyrCysHisGlyGluCysProPheProLeuAlaAspHisLeuAsnSerThrAsnHisAlaIleValGlnThrLeuValAsnSerValAsnSerLysIleProLysAlaCysCysValProThrGluLeuSerAlaIleSerMetLeuTyrLeuAspGluAsnGluLysValValLeuLysAsnTyrGlnAspMetValValGluGlyCysGlyCysArg

1. A process for recombinantly preparing a biologically active protein of the TGF-βsuperfamily, characterized in that (a) a protein, whose aminoterminus consists of the pro sequence of a protein of the TGF-β) superfamily, or parts thereof, to which the mature domain of this protein, or of another protein of the TGF-β superfamily which exhibits at least 35% homology with mature BMP-2, is attached, is expressed in prokaryotes under conditions in which at least a part of the protein is obtained in the form of inclusion bodies, (b) the inclusion bodies are isolated and solubilized under denaturing conditions, (c) the denatured, monomeric and biologically inactive protein, which has been solubilized from the inclusion bodies, is renatured, thereby enabling folding and dimerization to give the soluble, biologically active conformation, and (d) where appropriate, after the renaturation, the mature protein is proteolytically released from its pro form.
 2. The process as claimed in claim 1, characterized in that drBMP-2 is prepared.
 3. The process as claimed in one of the preceding claims, characterized in that for the purpose of increasing expression in prokaryotes, the proprotein is modified with an aminoterminal affinity label, in particular a polyhistidine sequence.
 4. The process as claimed in one of the preceding claims, characterized in that a pET vector, in particular pET-15b, is used as expression vector.
 5. The process as claimed in one of the preceding claims, characterized in that the codon choice in the expression construct is optimized for translation in the host organism.
 6. The process as claimed in one of the preceding claims, characterized in that tRNAs which are rare in the host organism and which are required for translating the expression construct are also overexpressed.
 7. The process as claimed in one of the preceding claims, characterized in that the overexpression is effected using the T7 expression system in BL21 (DE3) and in that the gene for the arginine tRNA (dnaY) which is rare in Escherichia coli is cotransformed with the expression construct.
 8. The process as claimed in one of the preceding claims, characterized in that the cells are disrupted by means of high pressure dispersion, enzymic lysis or ultrasonication.
 9. The process as claimed in one of the preceding claims, characterized in that the inclusion bodes are purified by washing with a detergent-containing solution, with the detergent concentration being selected such that interfering cell proteins and membrane constituents, but not the inclusion bodies, are solubilized.
 10. The process as claimed in the preceding claim, characterized in that the detergent is Triton X100.
 11. The process as claimed in one of the preceding claims, characterized in that chaotropic substances or combinations of chaotropic substances are used for solubilizing the inclusion bodies.
 12. The process as claimed in claim 11, characterized in that the chaotropic substance employed is guanidinium chloride at a concentration of from 3 to 8 M or urea at a concentration of from 6 to 10 M.
 13. The process as claimed in one of the preceding claims, characterized in that the solubilization takes place under reducing conditions.
 14. The process as claimed in claim 13, characterized in that the reducing agent employed is dithiothreitol and/or β-mercaptoethanol.
 15. The process as claimed in one of the preceding claims, characterized in that after the solubilization, the free cysteines are covalently modified.
 16. The process as claimed in one of claims 13 to 15, characterized in that after the solubilization, the reducing agent is removed.
 17. The process as claimed in one of the preceding claims, characterized in that after the solubilization, a preliminary purification takes places under denaturing conditions.
 18. The process as claimed in one of the preceding claims, characterized in that the proprotein is renatured by decreasing the concentration of the denaturing agent(s) down to a level which is not denaturing or is only weakly denaturing.
 19. The process as claimed in claim 18, characterized in that the concentration of the denaturing agent(s) is decreased by slowly diluting the solubilizate, continuously or stepwise, in renaturation buffer.
 20. The process as claimed in claim 19, characterized in that an intermixing which is as thorough as possible, for example by means of stirring, is effected during the dilution.
 21. The process as claimed in claim 18, characterized in that the concentration of the denaturing agent(s) is decreased by dialyzing the solubilizate against renaturation buffer.
 22. The process as claimed in one of the preceding claims, characterized in that use is made of a renaturation buffer having a neutral to alkaline pH.
 23. The process as claimed in claim 22, characterized in that the renaturation buffer contains L-arginine.
 24. The process as claimed in one of the preceding claims, characterized in that the renaturation buffer contains at least one thiol component in reduced and oxidized form.
 25. The process as claimed in claim 24, characterized in that the thiol component is GSH/GSSH.
 26. The process as claimed in one of the preceding claims, characterized in that after the renaturation process has been concluded, the sample is dialyzed against an acid buffer.
 27. The process as claimed in claim 26, characterized in that prior to the dialysis, the sample is reconcentrated against the acid buffer.
 28. The process as claimed in claim 26 or 27, characterized in that the constituents which are insoluble under acid conditions are separated off.
 29. The process as claimed in one of the preceding claims, characterized in that the pro form is purified chromatographically.
 30. The process as claimed in claim 29, characterized in that the purification is effected by means of RP-HPLC.
 31. The process as claimed in claim 29, characterized in that the purification is effected by means of heparin affinity chromatography.
 32. The process as claimed in one of the preceding claims, characterized in that the pro form is cleaved enzymically, by means of a protease, to give the biologically active form.
 33. The process as claimed in claim 32, characterized in that the protease employed is a prohormone convertase.
 34. The process as claimed in claim 32, characterized in that use is made of a protease which recognizes a dibasic sequence motif.
 35. The process as claimed in claim 32, characterized in that use is made of a protease which prefers a basic residue in the P1 position (immediately amino terminally to the cleavage site).
 36. The process as claimed in claim 35, characterized in that the cleavage is effected using trypsin.
 37. The process as claimed in one of claims 32 to 36, characterized in that at least one protease inhibitor is added for the purpose of limiting the proteolysis.
 38. The process as claimed in one of the preceding claims, characterized in that solubilizers which do not inhibit the enzyme are used to increase the solubility of the released protein at the pH at which the cleavage takes place.
 39. The process as claimed in claim 38, characterized in that the solubilizer employed is trishydroxymethyl-aminomethane and/or urea.
 40. The process as claimed in claim 39, characterized in that use is made of 0.1 to 2 molar trishydroxymethyl-aminomethane and/or 1 to 5 molar urea.
 41. The process as claimed in one of the preceding claims, characterized in that the mature or truncated mature protein is purified by means of precipitation and/or hydrophobic chromatography.
 42. The process as claimed in claim 41, characterized in that isoelectric precipitation is carried out and the precipitated protein is resolubilized in acid solution.
 43. The process as claimed in claim 41, characterized in that precipitation is effected by decreasing the solubilizer concentration.
 44. The process as claimed in one of claims 41 to 43, characterized in that precipitating conditions are established in the presence of chromatographic material.
 45. The use of the BMP-2 pro form obtained as claimed in claim 1 a to c for producing a pharmaceutical.
 46. The use as claimed in claim 45 for producing a pharmaceutical for promoting bone healing processes and bone neoformation processes.
 47. A proBMP-2 having the amino acid sequence Gly²⁰-Arg³⁹⁶ of Table 2 and identical sequences which lack a part of the pro sequence Gly²⁰-Arg ²⁸², which can be obtained using the process of claim
 1. 48. A composition for promoting bone growth, characterized by a content of proBMP-2 according to Table 2 having the complete or truncated pro sequence Gly²⁰-Arg²⁸² as active compound.
 49. A process for producing a composition for promoting bone growth, characterized in that proBMP-2 according to Table 2 having the complete or truncated pro sequence Gly²⁰-Arg²⁸² is formulated into a preparation which is suitable for administration.
 50. The process as claimed in claim 49, characterized in that the preparation comprises a physiologically tolerated support material which is coated or impregnated with the proBMP-2 active compound. 